Abstract
The soil bacterium Pseudomonas putida injects toxic proteins into neighbouring competitors, including resilient phytopathogens, using the Type VI secretion system (T6SS). The secretion of toxins endows P. putida with a significant fitness advantage, allowing this biocontrol agent to thrive in plant-related polymicrobial environments and prevent phytopathogen infections. Despite its agricultural significance, the toxin repertoire of P. putida, particularly those secreted via the K2- and K3-T6SSs, remains poorly understood. We present a comprehensive molecular study of Tke5, a potent toxin encoded within the K3-T6SS, which represents the initial biophysical and functional analysis of the BTH_I2691 family. Our data demonstrate that Tke5 is a pore-forming toxin that disrupts bacterial membranes through selective ion transport, inducing membrane depolarisation and cell death. Tke5 is neutralised by Tki5 in the inner membrane of Gram-negative bacteria. Unlike detergent-like pore-forming toxins, Tke5 preserves overall membrane integrity, avoiding large, non-specific disruptions. This mechanism offers a powerful approach to targeting resilient phytopathogens. This study reveals a previously undescribed mode of action within a widespread yet understudied toxin family. Our findings highlight the potential of P. putida as a biocontrol agent, offering alternatives to chemical pesticides by exploiting novel toxin mechanisms, crucial for developing effective strategies to combat plant pathogens.
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Data availability
The data are available within the manuscript. Supplementary Datasets 1–4 are available as additional supplementary files and contain: the list of proteins belonging to the BTH_I2691 family, the list of proteins identified by mass spectrometry from the Tke5 purified band, the numeric data for charts included within the article, and the flow cytometry raw data, respectively. Uncropped and unedited gel images are included in Supplementary Fig. 6. The gating strategy for all flow cytometry plots is included in Supplementary Fig. 7. The sequence of a newly generated vector available as a tool for the scientific community has been deposited into NCBI Genbank with accession number PQ628036 and into the SEVA repository https://seva-plasmids.com/.
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Acknowledgements
This publication is part of the project PID2021-123000OB-100, funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU. P.B. acknowledges the financial support received from the Spanish Minister of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) through the Ramón y Cajal Program (RYC2019-026551-I, ESF Investing in your future), the research grant from the State Subprogram for Knowledge Generation PID2021-123000OB-I00 and PID2024-159235OB-I00 (ERDF/EU), and the research grant from the State Subprogram for Promotion of Research Consolidation CNS2022-135585 (European Union NextGenerationEU/PRTR). D.A.-J. acknowledges support by the MICIU Contract PID2021-127816NB-I00, PID2024-155225NB-I00, and the Basque Government’s Department of Education IT1745-22. A.A. and M.Q.-M. acknowledge financial support by the Spanish Government MICIU/AEI/10.13039/501100011033/FEDER, UE (Project 2019-108434GB-I00 and Project PID2022-142795 NB-I00), Generalitat Valenciana (project CIGRIS/2021/021), and Universitat Jaume I (Project UJI-B2022-42). M.Q.-M acknowledges support from the Spanish Ministry of Science and Innovation (Project IJC2018-035283-I funded by MICIU/AEI/10.13039/501100011033) and Universitat Jaume I (project UJI-A2020-21). We thank Cayo Ramos, Emilia López Solanilla, Francisco M. Cazorla, Ehr Min Lai, María Milagros López, and Inmaculada Sampedro for their kind gifts of phytopathogenic strains.
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Conceptualisation: A.A., D.A.-J., P.B. Methodology: C.V., A.A.-R., M.Q.-M., J.A.-A., J.R.-P., A.F.-C., C.C.-N., A.R., J.P.-N., and C.C. Investigation: A.A., D.A.-J., and P.B. Visualisation: C.V., A.A.-R., M.Q.-M., and P.B. Supervision: M.Q.-M., A.G.-M., A.A.-R., A.A., D.A.-J., and P.B. Writing—original draft: A.A., D.A.-J., and P.B. Writing—review and editing: C.V., A.A.-R., and M.Q.-M.
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Velázquez, C., Arce-Rodríguez, A., Altuna-Alvarez, J. et al. Tke5 is a Pseudomonas putida toxin that kills plant pathogens by depolarising membranes. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09863-w
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DOI: https://doi.org/10.1038/s42003-026-09863-w
